Fixation is the first important step in preparing cell and tissue specimens for use in a wide range of analytical tests. Some exemplary tests include immunohistochemistry (IHC), flow immunocytometry, in situ hybridization (ISH) with nucleic acid probes, in situ polymerase chain reaction (PCR), and PCR. These tests are typically used to detect particular DNA or RNA sequences, peptides, proteins, or other kinds of biomolecules, drugs, and general analytes.
Fixation stabilizes microscopic cellular structures and compositions in the specimens to allow them to withstand subsequent processing and to preserve them for retrospective analyses. The fixed cell and tissue specimens can also be used to extract biosynthetic molecules for biochemical or nucleotide sequence analysis. Without fixation, it would be difficult, if not impossible, to sensitively detect, localize, and quantitate biosynthetic or environmental molecules in many kinds of cell and tissue specimens.
A good fixative should harden cell and tissue components to prevent decomposition, putrefaction, and autolysis. The physico-chemical process of tissue modification by a fixative is gradual and complex, involving diffusional penetration into the tissue and a variety of potential chemical reactions. To date, no ideal fixative has been found, i.e., a fixative that perfectly preserves cellular morphology and yet does not modify the specimen composition so as not to change the reactivity of the analyte species therein for subsequent detection. Because of this predicament, the selection of a particular fixative generally entails multiple considerations. Thus, there are many fixatives currently in use.
A fixative with a high content of alcohol or other organic solvent, particularly when acidified with a mild organic acid, hardens tissue specimens by precipitation and coagulation. Such a fixative has several advantages. First, since the fixative does not covalently modify the constituent molecules in the tissue, the reactivity of most antigens in the tissue toward antibodies remains very high. Second and for the same reason, nucleic acids in the tissue may be easily extracted in good condition. Third, the fixative may be completely flushed out of the tissue by rehydrating the tissue in a buffered solution.
Such a fixative, however, has one major drawback. The drawback is that the microscopic morphology of an alcohol- or other solvent-fixed tissue is not as detailed as that of a tissue fixed with a covalent-binding fixative.
In contrast to an alcohol- or other solvent-based fixative, a covalent-binding fixative, such as formaldehyde, provides excellent cellular preservation. Formaldehyde (CH.sub.2 O) was first reported as a tissue preservative by F. Blum in 1893 (10 Z. Wiss. Mikrosc. 314). It is now the most widely used fixative in histopathology because it is cheap, simple to use, and provides consistent results. A formulation of formaldehyde found in most U.S. and foreign research and clinical laboratories is neutral buffered formalin (NBF). It may also be called "buffered neutral formalin." See R. Lillie, Histopathologic Tech. 300 (1948). Other formaldehyde formulations include: 10% formalin; alcoholic formalin; calcium acetate formalin; Bouin's Fluid (containing picric acid or acetic acid, pH 1.6); Cajal's formalin-ammonium bromide; formalin/alcohol/acetic acid; paraformaldehyde (polymerized formaldehyde); and formol-saline (G. Clark, Staining Procedures 13-16 (1981)).
The commercially available saturated aqueous formaldehyde stock containing 10% methanol stabilizer is called formalin. Its formaldehyde concentration can be denoted in several ways. In particular, it can be denoted as a 100% saturated, a 37% w/w, a 40% w/v, or a 13.3 M solution. A usual working dilution of this stock for fixatives is 1:10 (initial:final) by volume. Such a dilution produces a 10% saturated solution which can also be denoted as 3.7% w/w, 4.0% w/v, or 1.3 M formaldehyde. For example, the standard NBF solution of the U.S. Armed Forces Institute of Pathology Formulation (AFIP) is a 1:10 v:v dilution of formalin in a phosphate buffer at a pH of 6.8 to 7.2 (Laboratory Methods in Histotechnology (eds. Edna Prophet et al., 1992)).
The chemistry of aqueous formaldehyde has been thoroughly reviewed, notably in J. Walker's treatise Formaldehyde, ch. 8 (1944). Formaldehyde is a gas that rapidly combines with water (&gt;99.9% according to C. H. Fox et al., 33 J. Histochem. Cytochem. 845-853 (1985)) to form the hydrate, methylene glycol: EQU CH.sub.2 O+H.sub.2 O.fwdarw.CH.sub.2 (OH).sub.2
Methylene glycol can be polymerized by commercial processes to form paraformaldehyde (polyoxymethylene glycol): EQU nCH.sub.2 (OH).sub.2.fwdarw.(CH.sub.2 O).sub.n.H.sub.2 O+(n-1)H.sub.2 O
which is sometimes used instead of formalin in the formulation of formaldehyde fixatives. In a neutral to alkaline buffered solution, paraformaldehyde depolymerizes to methylene glycol which dehydrates into an equilibrium with active carbonyl formaldehyde (Walker, supra, pp. 74-75). Thus, its effect on tissue is the same as diluted formalin.
Methylene glycol rapidly penetrates into tissue by diffusion at a rate that varies inversely with the tissue specimen temperature. Tissue penetration has been measured at 0.5 cm in 8 hours in rabbit liver (W. T. Dempster, 107 Am. J. Anat. 59-72 (1960)). Dehydration of methylene glycol within the tissue maintains an effective level of reactive carbonyl formaldehyde. Fixation reactions of carbonyl formaldehyde are much slower than the rate of penetration and are temperature dependent. Thus, when .sup.14 C formaldehyde was applied to semi-thin (16 .mu.m) tissue sections of rat kidney, the binding reaction took over 24 hours to reach equilibrium (C. H. Fox et al., 33 J. Histochem. Cytochem. 845-853 (1985)).
Formaldehyde, which is a very reactive electrophilic species, fixes tissue by combining with proteins and nucleic acids therein (Feldman, 13 Prog. Nucleic Acid Res. Mol. Biol. 1-49 (1973)). Formaldehyde modifications of the tissue proceed in two kinetically distinct stages. Initial reactions modify primary amines (lysine) and thiols (cysteine), and purine but not pyrimidine bases of nucleic acids, forming mono- and di-methylol derivatives, not Schiff bases. Regardless of whether the reaction involves nucleotides, nucleic acid polymers, amino acids, or proteins, this stage reaches equilibrium within 24 to 48 hours. These labile adducts are rapidly reversible if the formaldehyde is removed from the tissue.
Subsequent reactions involve the methylol derivatives that are covalently bound in the tissue. These secondary reactions form methylene crosslinks which are not reversible upon washing. In proteins, the secondary crosslinking reaction occurs via methylene bridges that join the first-modified sites to adjacent, less reactive functional groups including primary amides (glutamine, asparagine), guanidine groups (arginine), and tyrosine ring carbons (H. Fraenkel-Conrat & H. S. Olcott, 70 J. Am. Chem. Soc'y 2673 (1948)). This reaction is very gradual, accumulating over at least 30 days of fixation, and generates relatively stable covalent crosslinkages. In nucleic acids, secondary reactions also result in chain crosslinking. In addition to chain crosslinking, the reaction can produce crosslinkages between nucleic acids and proteins.
All of these secondary reactions produce a lattice of crosslinkages within and between macromolecules in the fixed tissue. The net effect of all covalent modifications is to partially denature the biopolymers in the tissue by interfering with the normal noncovalent bonding patterns of the charged protein side chains, and to lock the conformation into an inflexible configuration, i.e., the secondary nature of the macromolecules is not changed (Mason et al., 39 J. Histochem. Cytochem, 225 (1991)), while the conformation is locked into an inflexible configuration.
These secondary crosslinking reactions have an adverse impact on the analytical tests that are performed on the fixed tissue. For example, selective staining of a macromolecular species (protein, nucleic acid) or a smaller molecule (protein adduct, drug, metabolite, signal transduction species, lipid, etc.) in fixed tissue is often performed using an antibody that binds specifically and with high affinity to the analyte in the tissue. For sequence-specific detection of nucleic acids, a detectable complementary oligo- or poly-nucleotide sequence (probe) can be used for hybridization. Hybridization can be done on intact cell structures (in situ) for cytometric assay (e.g., by microscopy or flow cytometry). However, crosslinkages inside the fixed tissue prevent the large probe molecules employed in these tests, particularly antibodies and oligo- or polynucleotides, from penetrating. Reduced access by these probe molecules translates into loss of assay sensitivity.
Hybridization can also be done on soluble extracts prepared from tissue or cells for a composition assay (e.g., in gels or blots). Fixative modifications can compromise either the extraction efficiency, or the reactivity of the analyte. For example, fixation may affect the extraction efficiency of nucleic acids or the efficiency of subsequent nucleic acid amplification.
Similarly, one of the noncovalent binding forces that causes adhesion of the antibody or probe is the Coulombic attraction between opposite charges which produces hydrogen bonding, i.e., the pairing of oppositely charged groups on the test and analyte molecules. However, side-chain modifications and crosslinkages in the tissue can interfere with the capacity of an analyte to form these noncovalent bonds with an antibody or nucleic acid probe. Thus, it can be seen that formaldehyde modifications of the sulfhydryl and charged amino side chains that are involved in specific noncovalent binding interactions with the applied ligand, are deleterious to assay sensitivity.
A large target analyte such as a protein containing many potential epitopes can be detected using many different antibodies. Different epitopes have different polarity and sensitivity to formaldehyde fixation, presenting a range of susceptibilities to modification. There are many examples of target analytes that are detectable with one antibody, but not with another after extensive formaldehyde fixation. At one extreme, there are many antibodies that can only be used on frozen-sectioned tissue. Some of these can only tolerate a brief post-fixation of tissue in acid alcohol before antibody binding.
With many other antibodies, a progressive loss of antigenic reactivity has been found during prolonged formaldehyde fixation. The widely expressed cancer marker protein p53, for example, gradually loses all of its reactivity toward monoclonal antibody PAb1801 when fixed in formaldehyde for between 6 and 24 hours (R. Silvestrini et al., 87 J. Nat. Cancer Inst. 1020 (1995)). Similarly, the diagnostically important epithelial cell marker protein keratin gradually becomes unable to bind with a monoclonal anti-keratin antibody if the tissue is fixed in formaldehyde for up to 24 hours (H. Battifora & M. Kopinski, 34 J. Histochem. Cytochem. 1095-1100 (1986)).
There are other antibodies that are effective on tissue fixed for one day, but are less effective on tissue stored in NBF for longer periods. Some of these antibodies which are commonly employed in tumor diagnosis include lymphocyte antigens, vimentin, desmin, neurofilaments, cytokeratins, S100 protein, prostate specific antigen, thyroglobulin, and carcinoembryonic antigen (A. S. Leong & P. N. Gilham, 4 Pathology 266-268 (1989)). Other examples of such antibodies can be found in the biomedical literature.
Thus, it is important to control the tissue fixation time to achieve a compromise between the preservation of tissue morphology and the loss of antigenicity. As a general rule, the duration of aldehyde fixation should be kept to a minimum so as to allow the specimen to be tested using a wide range of different antibodies.
The quality and reproducibility of immuno-assay results also depend on the fixation time of the tissue. Currently, fixation is terminated by physically exchanging alcohol for the fixative solution. Leong and Gilham recommend fixing surgical histopathology specimens for no more than 6 hours. According to them, surgical pathology specimens are usually sampled after being fixed for 4 to 24 hours. However, in practice, the bulk of the surgical resection is often retained in formaldehyde for future resampling, which may occur after 3 or more days.
Autopsy specimens are usually fixed for between 3 and 14 days, depending on convenience. However, under some circumstances, it may not be expedient for laboratory personnel to closely monitor the specimens to achieve a preferred fixation time. These personnel may be occupied with other business and thus be absent when the fixative solution should be removed.
In addition, a biopsy or postmortem specimen that is not processed in-house may be sent to a pathology laboratory in a fixative. This transportation time often adds to the total fixation time of the specimen. Also, a specimen that arrives on Friday may not be processed until after the weekend, thereby extending the total fixation time even longer.
Moreover, a laboratory technician may accumulate many specimens for batch processing rather than separately process each sample after a predetermined fixation time. Thus, accurate records of the total fixation time of each individual sample may not be available for later reference for quality control standardization.
Variable staining is common when IHC is applied to archival paraffin-sections which were fixed for an unknown period of time. Such variations can be a "hidden variable" that confounds retrospective research experiments on the expression of putative cancer marker proteins that are fixation-sensitive such as the most widely expressed cancer marker protein p53 (P. Hall & D. Lane, 172 J. Pathol. 1-4 (1994)).
An important issue relating to histopathologic applications of immunologic and genetic tests is their reproducibility and quality. Recently, the College of American Pathologists and several other groups petitioned the Food and Drug Administration (FDA) to classify the nearly 2,500 antibodies sold in the U.S. as Class II medical devices (R. Stone, 268 Science 494 (1995)). The FDA has announced its intention to classify certain IHC reagents as Class II or Class III medical devices. This would require manufacturers to document the accuracy and precision of the tests (C. Graziano, 10 Col. Am. Pathologists 1 et seq. (1996)). Thus, both the FDA and the pathologists in the field recognize a need for better quality control and greater standardization of immunohistochemical procedures.
Fixation-induced loss of target epitopes can be compensated for by using several techniques, which have been termed antigen "retrieval," "restoration," "un-crosslinking," or "unmasking" in the literature. These techniques are performed on thin tissue sections which are individually processed. However, these techniques do not necessarily remedy the loss of standardization owing to uncontrolled variations in fixation and remediation. Variable fixation time requires a variable and unknown amount of uncrosslinking/unmasking to reach the same level of immunoreactivity, if in fact immunoreactivity is not irreversibly lost.
A number of antigens concealed by formaldehyde fixation can be re-exposed by applying protease solution to the tissue section (S. Huang et al., 35 Lab. Invest. 383 (1976); H. Battifora & M. Kopinski, 34 J. Histochem. Cytochem. 1095-1100 (1986)). However, more prolonged exposure to formaldehyde necessitated more vigorous proteolysis so as to recover a constant level of immunoreactivity. Proteolysis must be minimized because extensive protease digestion degrades the tissue morphology. In practice, the length of formaldehyde exposure could vary in different samples, suggesting the impracticality of standardizing the proteolysis time. Different antigens require different protease treatments. Therefore, this is not a simple way to address standardization concerns.
Microwave antigen retrieval was disclosed by M. E. Key, S. R. Shi, and K. L. Kalra in U.S. Pat. No. 5,244,787 (Sep. 14, 1993) and in S. R. Shi et al., 39 J. Histochem. Cytochem. 741-748 (1991). The method involves boiling the tissue section on a microscope slide in an aqueous solution selected from various defined pH and ionic compositions. Bankfalvi et al., 174 J. Pathol, 223-228 (1994) found that an equally effective and more expedient method was to autoclave hydrated sections. Recent developments of a variety of novel methodologies are summarized in a review by S. R. Shi, R. J. Cote & C. Taylor (45 J. Histochem. Cytochem. 327 (1997)), in which the need in this field for better optimization and standardization is stressed.
Cattoretti et al., 171 J. Pathol. 83-98 (1993) compared proteolysis to microwaving in various solutions. They found that some antigens benefit selectively from either one of the treatments, but not from both. The authors inferred that the common mechanism of antigen unmasking methods was related to protein denaturation. However, there was no obvious pattern relating the amino acid composition of particular protein epitopes to the deleterious effects of formaldehyde on their subsequent detectability by immunostaining with different monoclonal antibodies. Consistent with the findings of others in the field (S. R. Shi et al., 39 J. Histochem. Cytochem. 741-748 (1991); A. S. Leong & P. N. Gilham, 4 Pathology 266-268 (1989)), these authors found that some, but not all epitopes can be fully recovered after over-fixation, either by proteolysis or by heating. Because some fixation-sensitive antigens exist for which antigen retrieval is not effective, and because other fixation-sensitive antigens exist which may benefit from antigen retrieval only if they are overfixed, these differences give rise to a need in the art for a definitive method and composition that would standardize the extent of antigen masking by standardizing the fixation time.
DNA content can be measured in single cells in formalin fixed paraffin embedded tissues, or in cells recovered therefrom, to assay for cell proliferation by image analysis or flow cytometry. Formalin over-fixation interferes with the measurement of DNA content by decreasing the binding of propidium iodide and other fluorescent DNA-binding dyes, probably because it makes crosslinked DNA-histone complexes. Thermal antigen retrieval was applicable to this problem using cell suspensions or tissue sections (W. R. Overton & J. P. McCoy, 16 Cytometry 351-356 (1994); W. R. Overton & J. P. McCoy, 26 Cytometry 166-171 (1996)).
DNA or RNA can be extracted from formalin fixed, paraffin-embedded tissue samples for genetic analysis. These can be quantitated and then amplified using the PCR to determine the presence of a gene sequence such as for expression or mutation analysis. Formalin-fixed paraffin embedded tissue was only amenable to PCR sequence analysis if the tissue was fixed in NBF for approximately 12-24 hours (J. J. O'Leary et al., 26 Histochem. J. 337-346 (1994); C. E. Greer et al., 95 Am. J. Clin. Pathol. 117-124 (1991); F. Karlsen et al., 71 Lab. Invest. 604-611 (1994)). PCR in situ/in situ PCR methods are used for genetic analysis of archival, formalin-fixed paraffin embedded tissues, for example, to detect viruses and oncogene mutations in single cells. Limited fixation of 24-48 hours gave the best in situ amplification results (J. J. O'Leary et al., 26 Histochem. J. 337-346 (1994)). Thus, over-extended periods of fixation can adversely effect the results of these types of tests as well as others mentioned above.
Quantitative variations in tissue formaldehyde conjugation are difficult to assess retrospectively in IHC procedures. Battifora proposed that a partially fixation-sensitive antigen that is subject to a gradual loss of immunoreactivity in proportion to the extent of fixation in NBF could serve as an internal control. Battifora, in 96 Am. J. Clin. Pathol. 669-671 (1991), proposed to calibrate the average decrease of antibody binding in a fixed tissue by staining a section for a "universal surrogate epitope" for which he proposed using the ubiquitous endothelial marker vimentin. However, this method does not directly address the need for standardized fixation for total quality control.
In view of the above, there is a need in the art for a method and a composition that would prevent the secondary crosslinking reactions from occurring without the need to physically remove the cell or tissue specimen from the fixative solution or without the need to closely monitor the fixation time of the specimen or both. There is also a need in the art for a method and a composition that would allow for economical batch processing of cell or tissue specimens in a fixative while at the same time standardizing the fixation time of those specimens without the need for close supervision. There is a further need in the art for a method and a composition that would make analytical testing methods such as IHC and ISH more sensitive and consistent by raising the level of quality control as regards the tissue fixation time without a high economic cost.
Accordingly, it is an object of the present invention to provide for a method and a composition for preventing the secondary crosslinking reactions from occurring without the need to physically remove the tissue from the fixative solution or without the need to closely monitor the fixation time of the tissue specimen or both. It is a further object of the present invention to provide for a method and a composition that would allow for the economical batch processing of cell or tissue specimens in a fixative while at the same time standardizing the fixative time of those specimens without the need for close supervision. It is a further object of the present invention to provide for a method and a composition that would standardize an important variable in the fixation of tissue specimens, i.e., the time of fixation with formaldehyde.
These and other objects of the invention will be more readily understood by reference to the following summary, detailed description, and the appended drawings.